Capillary electrophoresis fluorescent detection system

ABSTRACT

The invention is a high sensitivity and high throughput capillary multiwavelength fluorescence detection system. The fluorescent detection system is configured to illuminate a relatively large volume of a single capillary or a plurality of capillaries, with a pixelated detection system capable of imaging an area of each capillary that differentiates the capillary walls, the space between the capillaries, and the internal liquid volume within the capillary. Only the desired pixels or image area are used for processing and generating an output signal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/121,043, filed Dec. 9, 2008, titled CapillaryElectrophoresis Fluorescent Detection System.

This application is a continuation-in-part of commonly assignedapplication Ser. No. 11/299,643 filed Dec. 12, 2005 entitled MultiplexedCE Fluorescence System.

FIELD OF THE INVENTION

This invention relates to capillary electrophoresis (CE) fluorescencedetection systems.

BACKGROUND OF THE INVENTION

Capillary electrophoresis (CE) instruments use electric fields toseparate molecules within narrow-bore capillaries (typically 20-100 μminternal diameter). CE techniques are employed in numerous applications,including DNA sequencing, nucleotide quantification, andmutation/polymorphism analysis. Samples analyzed by CE are oftendetected by fluorescence emission of the sample which has been taggedwith a fluorophore. The fluorophores are excited with a light source,and the intensities of the fluorescence emission represent theconcentration or amount of the sample components. Generally, the lightsource is focused on a narrow point on the sample to maximize the energyavailable for the excitation of fluorophore within the illuminatedvolume. The detector, which is usually a photomultiplier, photodiode,diode array, or CCD, is positioned to capture the maximum amount oflight from the sample, without specific discrimination of the capillarywalls or the background.

SUMMARY OF THE INVENTION

Embodiments of the invention provide a high sensitivity and highthroughput capillary electrophoresis multiwavelength florescencedetection system. The fluorescent detection system is configured toilluminate a single capillary or a plurality of capillaries, with apixelated detection system capable of imaging an area of each capillarythat differentiates the capillary walls, the space between thecapillaries, and the internal liquid volume within the capillary. Thedetector is coupled to a computer processing system capable of selectingpixels or areas of the image to process (e.g., integrate). The pixels orimage area is selected such that only fluorescent light from theinternal volume of the capillary, without light from the capillary wallsor background light from between the capillaries, is integrated. Thisresults in a larger signal to noise ratio relative to methods thatintegrate the light from the entire capillary cross-section. The systemis configured so that a width of at least one pixel defines the middleliquid volume of each capillary.

Embodiments of the invention also illuminate a relatively large volumeof the capillary to maximize the number of fluorophores available forexcitation within the illuminated area. This allows for a larger signalto noise ratio relative to methods that integrate light from only anarrowly focused point on the capillary. Further, since embodiments ofthe invention are able to differentiate between the capillary walls, thespace between the capillaries, and the internal liquid volume within thecapillary, the computer processing system can process and display thedata from the internal liquid volume and exclude the data from thecapillary walls and space between the capillaries to provide anexcellent signal output even though the large beam area may illuminatecapillary walls and space between capillaries.

Some embodiments of the invention include more than one detection windowto detect fluorophore emissions at different wavelengths. This allowsfor detection of multiple compounds within the same column. For example,a series of unknown DNA strands can be labeled with Fluorophore X, whilea known standard ladder of DNA strands with known molecular weights canbe labeled with Fluorophore Y. The use of multiple detection windows(coupled with multiple light sources), allows for the independentmeasure of the unknown DNA samples with known standards in a singlecapillary. This eliminates the need to run a standard compound andunknown compound in two separate capillaries.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a partially exploded schematic diagram illustrating a largevolume multi-wavelength fluorescence detection system for multiplexedcapillary electrophoresis in accordance with an embodiment of theinvention.

FIG. 2 is a schematic diagram illustrating an optical fiber bundle inaccordance with an embodiment of the invention.

FIG. 3A is a close up view illustrating an optical path in accordancewith an embodiment of the invention.

FIG. 3B is a close up view illustrating the optical path in accordancewith an embodiment of the invention.

FIG. 4 is a schematic of a CCD array image showing a differentiation ofinternal capillary volume, liquid capillary walls, and space between thecapillaries.

FIG. 5 is a picture of an imaged capillary array, showing a cleardifferentiation of internal capillary liquid, capillary walls, and spacebetween the capillaries.

FIG. 6A is an enlarged view of one of the capillaries shown in FIG. 5.

FIG. 6B is a capillary electropherogram trace that represents thesummation of all pixels in each column from FIG. 6A.

FIG. 7 is a series of electropherograms of single stranded DNA thatrepresents the summation of all pixels in each column from edge to edgeof the capillary shown in FIG. 6.

FIG. 8 is an electropherogram constructed from the middle 6electropherograms from FIG. 7 (i.e. excluding light from the walls ofthe capillary).

FIG. 9 is an electropherogram measured with large volume fluorescencedetection as discussed in FIG. 8 (lower trace) compared with anelectropherogram using a small volume fluorescence detection (toptrace).

FIG. 10 is a double stranded DNA electropherogram with single wavelengthlarge volume fluorescence detection as discussed in Example 1, whereineach separate electropherogram is from a separate capillary.

FIG. 11 is a carbohydrate (MALTRIN® M-200) separation electropherogramwith single wavelength large volume fluorescence detection as discussedin Example 2.

FIG. 12 is two electropherograms (one for each wavelength) generatedwith a two wavelength large volume fluorescence detection as discussedin Example 3.

DETAILED DESCRIPTION OF THE INVENTION

For the purpose of promoting an understanding of the principles of theinvention, reference will now be made to the embodiments illustrated inthe drawings and specific language will be used to describe the same. Itwill, nevertheless, be understood that no limitation of the scope of theinvention is thereby intended; any alterations and further modificationsof the described or illustrated embodiments, and any furtherapplications of the principles of the invention as illustrated therein,are contemplated as would normally occur to one skilled in the art towhich the invention relates.

In some embodiments, the invention includes a fluorescence detectionsystem. The detection system includes a sample vessel (e.g., acapillary) in which a sample is placed. A light source is included toemit light to excite a fluorescently labeled sample, and the system isconfigured to direct light on the sample vessel (sometimes referred toherein as a “detection window”) to illuminate a volume of more thanabout 100 micrometers×πr², where r is one-half of the inner diameter ofthe sample vessel or capillary. Although the light is intended toilluminate the internal capillary volume, it will also necessarily atleast partially illuminate the capillary wall and the space betweencapillaries. The problem of inadvertent illumination degrades thequality of the output signal, and this problem is exacerbated by therelatively large detection window as described herein.

Embodiments of the invention also include a fluorescence detectorcapable of imaging the entire cross section of the capillary (ormultiple capillaries), and has the resolution to allow it to clearlydifferentiate between the capillary wall, the internal capillary volume,and the space between capillaries. The detector is positioned to detectthe fluorescent emissions of the sample. The detector has the resolutionto image distinct parts of the image. For example, the detector can haveat least one pixel defining the internal volume of each capillary, atleast one pixel defining each capillary wall, and at least one pixeldefining the space between the capillaries. Any suitable detector may beused. However, detectors such as charge coupled devices (CCDs) areparticularly useful with embodiments of the invention. An example ofsuch a CCD is made by Starlight Xpress Ltd., model #: SXVR-H9, equippedwith an ICX285 CCD chip with 1392×1040 pixels in a two-third inch formatinterline camera and a pixel size of 6.45 μm×6.45 μm.

The detector is attached to a computer system or processor 111 capableof selecting the pixels for the final detection of fluorescentlight-whereby only the pixels corresponding to the internal capillaryvolume are chosen. Pixels corresponding to the capillary walls or thespace between capillaries are excluded from the final fluorescentsignal. In some embodiments, after the detector (e.g., CCD) records theimages, the processor calculates the time lapsed signal to noise ratioof the pixels along the x-axis. The capillary walls always have a lowersignal to noise ratio than the illuminated internal volume of thecapillary and the space between the capillaries has no signal.Accordingly, the processor (e.g., with software) can use these uniquecharacteristics of each region to define the regions. For example, thesedata discrimination and analysis functions can be written on Labviewversion 7.0 form National Instruments run on a personal computer.Accordingly, embodiments of the invention are useful for illuminating arelatively large volume of a fluorescently labeled sample, whileexcluding stray light from the capillary walls and light from betweenthe capillaries, thereby increasing the signal-to-noise ratio of theilluminated volume to provide a higher quality output.

The fluorescence excitation light source can be a gas discharge lamp(mercury or xenon), a laser (gas, solid state, dye, or semiconductor) ora light-emitting-diode (LED). In some embodiments the detection systemincludes non-coherent light sources as the excitation light source. Insome embodiments, the light source is a high power LED, which operatesat a current rating of least 100 milliAmps, preferably at 500 milliAmps,and even more preferably 700 to 1000 milliAmps.

An optical fiber bundle can be provided to direct the emitted light fromthe light source to the sample vessel detection window without focusingthe irradiation light. A large volume of each sample vessel isilluminated due to the non-focused illumination. In some embodiments,the detection window includes a bandpass filter for specific wavelengthdetection. A fluorescence detector capable of imaging the entirecapillary cross-section, with a differentiation of the walls of thecapillary, the internal capillary volume, and the space between thecapillaries, such as a CCD, is positioned to detect the fluorescentemissions of the sample. In addition, the use of the optical fiberbundle allows the illumination of multiple sample vessels simultaneouslyin multi-channel systems and the detector can monitor fluorescencesignals of multiple channels.

FIG. 1 illustrates a fluorescent detection system 10 in accordance withan embodiment of the invention. The embodiment of FIG. 1 includes amultiplexed capillary array electrophoresis system with a capillaryarray 20 and a high power LED 30 for large volume illuminationfluorescence detection. A “high power” LED is one which operates at acurrent rating of least 100 milliAmps, preferably at 500 milliAmps, andeven more preferably 700 to 1000 milliAmps. As shown, the high powerLED's light output is coupled to an optical fiber bundle 40 throughoptical couplers 50. In the embodiment shown, the fiber bundle lightentrance end 60 (i.e., proximal end) has a round shape to match theoutput of the LED while the exit end 70 (i.e., distal end) has arectangular shape with the long side having similar or larger dimensionthan the detection window on the multiple capillaries, as shown in FIG.2. In some embodiments, the optical fiber bundles include about 16,600optical fibers. Each optical fiber can be about 50 μm in diameter withnumeric aperture (N.A.) of about 0.55. The light entrance end can have adiameter of about 7.11 mm to match the optical coupler output area. Theexit end of the optical fiber can have dimensions of about 1.5 mm×about25.4 mm. With this dimension, roughly 30×510 optical fibers are packedinto a rectangular shape at the distal end of the optical fiber bundle.In addition, the exit end of the fiber bundle can be positioned at anangle relative to longitudinal axis of the capillary (e.g., betweenabout 30 degrees and 60 degrees, such as about 45 degrees). The anglehelps to eliminate the direct irradiation of the excitation light ontothe camera lenses to eliminate background noise.

As shown, filters 80 (which can be the same or dissimilar for eachother) can be included to block off unwanted excitation wavelengths fromthe LEDs. Filters 90 (which can be the same or dissimilar for eachother) can be used to select the desired fluorescent wavelength fordetection. Also as shown, a camera lens 100 can be used to collect thefluorescent emission from the detection windows of the multiplecapillaries while a two-dimensional detector such as a CCD 110 can beused to monitor the fluorescent emission. A processor, which would beconnected to the CCD to process the output from the CCD (e.g.,differentiate the regions and provide an integrated output signal), isnot shown.

Embodiments of the invention include configuring the CCD array in such away as to enable differentiation of the light coming from the capillarywalls, the internal capillary volume, and the space between eachcapillary. This allows one to select and detect light only from theinternal volume of each capillary. A CCD with a two-dimensional arrayarea of 1392 by 1040 pixels is preferred for imaging from about 1 up toabout 96 capillaries, while enabling differentiation of the walls of thecapillary, the internal capillary volume, and the space between thecapillaries.

The capillary array electrophoresis system shown in FIG. 1 has capillarywindows arranged on the same plane at the detection region tosimultaneously illuminate the detection window for on-column detection.During use, the capillary array has both ends immersed into buffersolution in which a high voltage is applied for electrophoresisseparation. The ends of the capillary array may be separated forindividual sample loading.

FIGS. 3A and B illustrate close up sections of detection windows inaccordance with embodiments of the invention. As shown, the exit end ofthe optical fiber bundle is positioned less than 1 mm away from thecapillaries and at about 45° against the capillaries. The light exitseach fiber of the optical fiber bundle with a divergence angle based onthe numeric aperture (N.A.) of the optical fiber. For example, anoptical fiber has a N.A. of 0.25 will have a divergence angle of about29° while a N.A. of 0.66 will have divergence angle of about 83°. Whenthe optical fiber bundle is positioned 1 mm or less from the capillarytubing window, approximate 2 mm of the tubing length will be illuminatedby the exit light from the optical fiber bundle. In addition, eachcapillary's detection window will be illuminated by more than oneoptical fiber from the optical fiber bundle.

Further, in some embodiments, the optical fiber position from the lightentrance and exit are randomized. In such embodiments, the uneven lightdistribution from the LED output is homogenized at the exit end of theoptical fiber bundle, which provides more consistent illumination to thesample volume.

Typical fluorescent detection systems focus the light source onto thesample with as small an area as possible. Fluorescent signal intensityis proportional to the incident light power and the amount offluorophore molecules present in the irradiation volume. Capillariesgenerally have an internal diameter of about 100 um or less, andfluorescence detection systems for HPLC or capillary electrophoresissystem generally focus the light source to a point much less than 100um. Focusing the light source increases the power density of incidentlight at the small detection volume. Therefore, more photons areavailable to excite the sample molecules within the small detection zone(<100 μm×πr², where r is the ½ of the radius inner tubing of thecapillary). Further, focusing the light source into a small areamaintains high resolution of separation. If the CE separation resolutionis smaller than the illumination area, the detection resolution lost.However, in most of multiplexed capillary electrophoresis applications,the separation resolution does not require the tight focusing (<<100um).

Therefore, instead of focusing the light to a small volume to obtainhigh power density for illumination, embodiments of the invention use ahigh power LED to provide high photon flux to illuminate a relativelylarge volume in which more molecules are excited to fluorescence becausemore sample molecules are available for excitation. In some embodiments,the system is configured to direct light on the sample vessel toilluminate a volume of more than about 50 micrometers×πr², where r isone-half of the inner diameter of the sample vessel. In otherembodiments, the system is configured to direct light on the samplevessel to illuminate a volume of more than about 500 micrometers×πr². Inyet other embodiments, the system is configured to direct light on thesample vessel to illuminate a volume of more than about 1,000micrometers×πr². In certain embodiments, the system is configured todirect light on the sample vessel to illuminate a volume of more thanabout 1,500 micrometers×πr². In some embodiments, the system isconfigured to direct light on the sample vessel to illuminate a volumeof less than about 2,000 micrometers×πr². In certain embodiments, thesystem is configured to direct light on the sample vessel to illuminatea volume of about 2,000 micrometers×πr².

As shown in FIG. 4, the CCD detector should be configured to detectthree regions of the capillary (for a single capillary) or capillaryarray (for multiple capillaries). In FIG. 4, region A includes the wallof each capillary, region B includes the internal volume of eachcapillary, and region C includes the space between capillaries. Eachregion should correspond to at least one distinct pixel in the detector.In some embodiments, region B (of each capillary) has at least 3 pixels.In other embodiments, region B (of each capillary) has at least 5pixels.

The height of the entire capillary array image may range from 1 pixel upto the y-axis length (in pixels) of the CCD. For example, A CCD arraywith width of 1392 pixels and length (y-axis) of 1040 pixels may be usedto image a 12-capillary system wherein the internal liquid volume width(x-axis) of each capillary is at least 6 pixels, the walls of eachcapillary (x-axis) is at least 1-pixel, and the space between eachcapillary is at least 20 pixels. The height of each image is at least 60pixels, but may be up to 1040 pixels, depending on how the capillaryimage is focused onto the CCD window. FIG. 5 shows an imaged capillaryarray, in which the internal volume of each capillary is about 6 pixels,each capillary wall is 2 pixels, and the space between capillaries isabout 80 pixels. The length of the capillary image is about 60 pixels. ACCD detector with 1392 by 1040 pixels was used to capture this image.FIG. 6 shows an enlarged view of one of the capillaries from FIG. 5. Thetwo vertical lines represent the wall of the capillary. The uneven lightdistribution from the capillary is due to scattering from imperfectsurface, dust, and incomplete removal of polyimide coating. FIG. 6Bshows the one dimensional display when summing all vertical pixelsintensities together for each column.

FIG. 7 shows the electropherograms of single stranded DNA separation. Asample similar to that illustrated in example 3 was used here fordemonstration. Only one LED and fiber optical bundle was used for theexcitation. A LED with 470 nm emission was used for the excitation whilemonitoring the fluorescence signal through a band pass filter withbandwidth from 500 nm to 550 nm. The samples were injected into one endof the capillaries by applying 5 kV for 20 seconds. After the sampleinjection, the injection ends of the capillaries were immersed intobuffers for separation under 150 V/cm separation field strength. Thefluorescent signal was recorded by a CCD capable of imaging the entirecross-section of the capillary, including the capillary walls, thevolume within each capillary, and the space between each capillary. Eachelectropherogram represents the time lapse signal from the summation ofall vertical pixels signal of each column (vertical line to verticalline) shown in FIG. 6A. The top trace to the bottom trace in FIG. 7shows 10 electropherograms that represent each column's (i.e. a columnwith a width of 1 pixel) signal during the time of electrophoresisseparation. The top trace is the left edge of the capillary, and thebottom trace is the right edge of the capillary. FIG. 7 indicates thatthe capillary image had two pixels for each side of wall withsignificantly lower S/N and 6 pixels for the internal volume with highS/N. An average of all 10 electropherograms, including the wall, resultsin a lower signal to noise ratio than an average of the middle 8electropherograms. To obtain a higher signal to noise ratio the wall'ssignal should be excluded and only the internal volume signal used toconstruct the final electropherogram. FIG. 8 shows the finalelectropherogram excluding the signal from the capillary's wall.

With the same irradiance, large volume fluorescence detection providesbetter S/N compared to small volume fluorescence detection since moresample molecules are available for detection. In FIG. 9, the bottomelectropherogram was constructed using large volume fluorescencedetection, in which a 1.5 mm section of capillary was illuminated; whilethe top electropherogram represented the small volume fluorescencedetection, in which only a 50 μm section of capillary was illuminated.The electropherogram has much better S/N for large volume fluorescencedetection (lower trace) than the small volume fluorescence detection(top trace).

In addition, as shown in FIG. 1, embodiments of the invention providefor multi-wavelength excitation and fluorescent detection. If the samplehas been labeled with two different fluorophores, it may require twodifferent wavelengths for excitation because of the absorptioncoefficiency is different for different fluorophores. Embodiments of theinvention provide for multiple wavelength excitation and detection withthe use of LEDs at different wavelengths to irradiate at differentlocations for different wavelength excitation and detection.

In such embodiments, as shown in FIG. 1, multiple light sources andoptical fiber bundles are used for excitation at multiple detectionwindows to excite the fluorophore at multiple wavelengths. In someembodiments, each detection window comprises a bandpass filter forspecific wavelength detection. All detection windows can be monitoredsimultaneously with a two-dimensional detector such as a charged coupleddevice (CCD) capable of imaging the entire cross-section of thecapillaries, with a clear pixelated differentiation of the capillarywalls, the internal capillary volume, and the space between thecapillaries, as described in detail above. When coupled with a computersystem capable of selecting individual pixels for integration, such thatpixels corresponding to the walls of the capillary are excluded fromintegration, this allows for an increase in the signal-to-noise ratio.When multiple detection windows are used, multiwavelength fluorescencesignals from the same separation sample vessel are obtained. Inaddition, the use of optical fiber bundle allows the illumination ofmultiple sample vessels simultaneously in multi-channel systems and thetwo-dimensional detector can monitor multiwavelength fluorescencesignals of multiple channels.

EXAMPLES

The examples below are merely illustrative and are not intended to limitthe scope of the invention.

Example 1 Double Stranded DNA Electropherogram and a Large VolumeDetection System

FIG. 10 shows the electrophoretic pattern of double-stranded DNA 100b.p. ladders obtained by an embodiment of the invention. This wasmeasured in a multicapillary system, and each trace represents adifferent capillary. In this example only one LED light source and fiberoptical bundle was used for the fluorophore excitation. The LED emittedat 470 nm for excitation while a band pass filter transmitted from 500nm to 600 nm was used to select the desire fluorescent wavelength fordetection. The separation matrix contained SYBR Green dye. Whendouble-stranded DNA binds to SYBR Green, the resulting DNA dye complexabsorbs the LED light and fluoresces at 522 nm. The samples wereinjected into one end of the capillaries by applying 5 kV for 5 seconds.After the sample injection, the injection ends of the capillaries wereimmersed into buffers for separation under 150 V/cm of a constantelectric field. The volume of illuminated liquid was 1500micrometers×πr², where r is one-half of the inner diameter of the samplevessel, which was 75 um.

Example 2 Carbohydrate Separation Electropherogram with Large VolumeFluorescence Detection

FIG. 11 shows the high resolution oligosaccharide profiling byelectrophoretic separation of carbohydrate MALTRIN® M-200 labeled with8-aminopyrene-1,3,6-trisulfonate (APTS) using an embodiment of theinvention. In this example only one LED and fiber optical bundle wasused for the excitation. A LED with 470 nm emission was used for theexcitation while monitoring the fluorescence signal through a band passfilter with bandwidth from 500 nm to 600 nm. The volume of liquidilluminated was 1500 micrometers×πr², where r is one-half of the innerdiameter of the sample vessel, which was 75 um. The samples wereinjected into one end of the capillaries by applying 5 kV for 10seconds. After the sample injection, the injection ends of thecapillaries were immersed into buffers for separation under 300 V/cm ofa constant electric field. The fluorescent signal was recorded by theCCD. MALTRIN® M-200 is a maltooligosaccharide ladder which contains atleast 16 individual oligomers as shown in FIG. 11.

Example 3 Multiple Wavelength Detection

The Staphylococcus aureus tuf gene has the following DNA sequence:

5′-TATTCTCAATCACTGGTCGTGGTACTGTTGCTACAGGCCGTGTTGAA3′-ATAAGAGTTAGTGACCAGCACCATGACAACGATGTCCGGCACAACTTCGTGGTCAAATCAAAGTTGGTGAAGAAGTTGAAATCATCGGTTTACATGAGCACCAGTTTAGTTTCAACCACTTCTTCAACTTTAGTAGCCAAATGTACTCACATCTAAAACAACTGTTACAGGTGTTGAAATGTTCCGTAAATTATTAGGTGTAGATTTTGTTGACAATGTCCACAACTTTACAAGGCATTTAATAATC ACTACGCTGAAGCT-3′TGATGCGACTTCGA-5′

The following DNA sequences were selected as primers for PCRamplification:

5′-TATTCTCAATCACTGGTCGT-3′ 5′-AGCTTCAGCGTAGTCTA-3′.

5′-TATTCTCAATCACTGGTCGT-3′ was labeled with a fluorescence dye (FAM) inthe 5′ position, and 5′-AGCTTCAGCGTAGTCTA-3′ was labeled with adifferent fluorescence dye (Cy-5) at the 5′ position. After the PCRamplification for Staphylococcus aureus DNA, one strand of PCR productcontained a green fluorescence dye while other strand of DNA contained ared fluorescence dye. After purification of the PCR product, 80% ofN-methylformamide was used to cleave the DNA at 110° C. for 30 minutes.Samples were then separated with electrophoresis without furtherpurification.

A capillary fluorescent detection system in accordance with theinvention was used to simultaneously separate and detect the fragmentsthat labeled with the different dyes. The embodiment of the inventionshown in FIG. 1 was used for the separation and detection. FIG. 12 showsthe electropherograms obtained with the present two wavelength (i.e.,colors) detection system. The bottom trace represents the emissionwavelength from 500 nm to 550 nm with excitation at 470 nm while the toptrace represents the emission wavelength from 620 nm and up with theexcitation at 560 nm. The volume of illuminated liquid was 1500micrometers×πr², where r is one-half of the inner diameter of the samplevessel, which was 75 um. The 480 nm to 550 nm represents the emissionfrom FAM labeled DNA fragments while the other wavelength represents theemission from Cy-5 labeled DNA fragments.

While the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives,modifications, and variations will be apparent to those skilled in theart in light of the foregoing description. Accordingly, it is intendedto embrace all such alternatives, modifications, and variations, whichfall within the spirit and broad scope of the invention.

What is claimed is:
 1. A fluorescence detection system, comprising: asample vessel comprised of capillaries; an LED light source to emitlight to excite a fluorescently labeled sample contained in saidcapillaries, the system configured to direct light on the sample vesselto illuminate the sample; a CCD fluorescence detector positioned todetect the fluorescent emissions of the sample, said fluorescencedetector capable of producing an image, having sufficient resolution todetect three distinct regions of the image, including a wall of eachcapillary, an internal volume of each capillary, and a space between thecapillaries, each region being defined by at least one pixel, and aprocessor capable of providing an integrated signal for selecting whichpixels to process for an integration of light intensity, with pixelsfrom the walls of the capillary and pixels from the space between thecapillaries specifically excluded from the integrated signal; andintegrating of the light intensity of said pixels to provide afluorescent signal.
 2. The fluorescent detection system of claim 1,wherein said sample vessel of said system contains at least two separatewindows, and the detection system includes at least two light sources,and the fluorescent light from each of the said two separate windows isimaged onto at least two separate areas of the CCD.
 3. The fluorescencedetection system of claim 1, wherein the system is configured to directlight on more than one sample vessel.
 4. The system of claim 1configured so that the width of at least one pixel defines the volume ofeach capillary.
 5. The system of claim 1 that uses multiple detectionwindows.
 6. The CCD fluorescence detection system of claim 1, with saidsystem being configured to detect fluorescent emissions at more than onewavelength.
 7. A method of detecting a fluorescence emission in a samplethe method comprising: introducing a fluorescently labeled sample into asample vessel; directing a light from an LED light source to excite thefluorescently labeled sample, the light directed on the sample vessel toilluminate the sample vessel; and detecting a fluorescent emission fromthe fluorescently labeled sample with a CCD fluorescence detectorcapable of producing an image having sufficient resolution to detectthree distinct regions of the image; said three regions including a wallof each capillary, an internal volume of each capillary, and a spacebetween the capillaries, each region being defined by at least onepixel, and using a processor capable of providing an integrated signalfor selecting which pixels to process for an integration of lightintensity, with pixels from the walls of the capillary and pixels fromthe space between the capillaries specifically excluded from theintegrated signal.